Nucleic acid amplification methods, especially Polymerase Chain Reaction (PCR) methods, are techniques of conveniently amplifying a desired nucleic acid fragment in vitro, which have become indispensable experimental methods in wide range of fields of biology, medicine, agriculture, and the like. In addition, a method of accurately quantifying DNA that serves as a template is developed by measuring nucleic acid amplification by PCR with the passage of time utilizing, for example, an intercalating dye (Non-Patent Publication 1). This method is referred to as a real-time PCR method, in the sense of distinguishing with conventionally known quantification PCR.
As the real-time PCR method, aside from the method using an intercalating dye, a method using a FRET (fluorescence resonance energy transfer) oligonucleotide probe has been known. As real-time PCR using the FRET oligonucleotide probe, a method utilizing 5′→3′ exonuclease activity of DNA polymerase (Taq Man Probe method), a method using Molecular Beacon probe, a method using two kinds of oligonucleotide probes designed to allow FRET to take place when hybridized to a target nucleic acid (HybriProbe method), and a method utilizing a FRET oligonucleotide probe containing ribonucleotide and a thermostable ribonuclease H (RNase H) (CycleavePCR method, Patent Publication 1), or the like have been known.
PCR methods are also applied to a method for detecting RNA, which is referred to as a Reverse Transcriptase PCR (RT-PCR) method. RT-PCR method is a method of synthesizing a DNA (cDNA) complementary to RNA using a reverse transcriptase having RNA-dependent DNA polymerase activity, in other words, reverse transcription activity, or DNA polymerase also having reverse transcription activity, and subsequently carrying out PCR using this cDNA as a template, thereby specifically amplifying and detecting cDNA derived from RNA. RT-PCR method is not only utilized in cloning of cDNA derived from mRNA or preparation of cDNA library, but also useful as a method of examining an expression state of a specified mRNA. Also, a real-time RT-PCR method in which RT-PCR is applied to real-time PCR enables accurate quantification of mRNA, so that the method is applied to expression analysis of genes or generation of expression profiles.
In RT-PCR, cDNA after the reverse transcription reaction forms a DNA/RNA hybrid with RNA that serves as a template for a reverse transcriptase. Therefore, from the beginning of the development of RT-PCR, cDNA is preferably made into a single strand before PCR by a treatment with an alkali, heat, or an enzyme after the reverse transcription reaction, in order to improve the reactivity of RT-PCR. For example, in a case where RNA that serves as a template has a high GC content, there is a report that it is effective to carry out an enzymatic treatment with ribonuclease H derived from Escherichia coli after the reverse transcription reaction (Non-Patent Publication 2).                Patent Publication 1: WO 2003/074696        Non-Patent Publication 1: Biotechnology (NY). 1993 September; 11(9): 1026-1030.        Non-Patent Publication 2: Biosci Biotechnol Biochem. 2003 November; 67(11): 2474-2476.        